Pulmonary arterial hypertension (PAH) is a rare lung vascular disease characterized by complex arterial remodeling that leads to narrowing and obliteration of the distal lung arterial bed, progressive increases in pulmonary arterial pressure and resistance, and ultimately right heart failure and death (1). PAH is triggered by endothelial cell (EC) injury and apoptosis (2). EC apoptosis and loss can result directly in distal arteriolar pruning and indirectly induces reactive EC proliferation to repair and regenerate the damaged lung microcirculation (3–5). With ongoing endothelial stress, an imbalance between endothelial injury and repair likely sets the stage for maladaptive arterial remodeling and vascular inflammation which are both typical pathological features of PAH (2). Senescence is a cellular state characterized by irreversible cell cycle arrest and the secretion of a pro-inflammatory and tissue-remodeling factors called the senescence-associated secretory phenotype (SASP) (6, 7). Accumulation of senescent cells can lead to altered inflammatory state and cellular senescence has been recognized as a promising target in the treatment of a number of pulmonary and cardiovascular diseases (8, 9). In PAH, senescent cells, including smooth muscle and ECs, are mainly localized to remodeled arteries and have been implicated in the progression from reversible to irreversible disease (10). However, interventions targeting cellular senescence in PAH have produced conflicting results (10–12), either greatly improving arterial remodeling and pulmonary hemodynamics or exacerbating the progression of disease, depending on the models studied and the timing of the intervention.

In this review we will explore the possible role of EC senescence in lung microvascular repair at early stages of PAH as well as its contribution in promoting vascular inflammation and remodeling in more advanced disease. We will also address the potential pitfalls in the use of senotherapeutic agents and discuss alternative strategies to target the accumulation of senescent cells in PAH. Further exploration of the role of senescence in the pathobiology of PAH may point to new novel treatment strategies to target cellular senescence, with the goal of reducing PAH progression, mortality, and reversing the disease.

Pulmonary hypertension (PH) represents a range of heterogeneous conditions which exhibit elevated pulmonary artery pressure (mean > 20 mmHg) and are classified into five separate groups based underlying etiology and clinical management approaches (13). Pulmonary arterial hypertension (PAH), or group 1 pulmonary hypertension (PH), is a rare and often fatal condition resulting from narrowing or loss of the precapillary arterial bed as defined hemodynamically by a normal pulmonary wedge pressure (mean < 15 mmHg) and high pulmonary vascular resistance (mean > 3 Wood units) (14). There are a number of clinical subtypes of PAH, including: idiopathic PAH; PAH associated with another disease, typically connective tissue diseases and congenital heart diseases (CHD) with large systemic to pulmonary arterial shunts; and heritable (H) PAH, which is most commonly caused by mutations in the bone morphogenetic protein receptor type II (Bmpr2) gene. While the pathogenesis of PAH is complex, it is generally accepted that EC injury and/or apoptosis caused by toxins, inflammation, or other noxious stimuli represents a triggering mechanism in the context of a genetic or other predisposition. This can result in EC dysfunction, increased vasoconstriction and ultimately pulmonary arterial remodeling and inflammation affecting both larger arteries and small arterioles (2), including the development of complex arterial remodeling, which is a hallmark feature of PAH (15). Although endothelial dysfunction and simple medial arterial thickening have been seen in all forms of PH, PAH is characterized by intimal lesions resulting in luminal encroachment and occlusion and often associated with adventitial remodeling and inflammation. The resulting complex arterial remodeling has been variously termed, ‘plexiform’ or ‘angioproliferative’, and is usually seen at branching points of the pulmonary arteries (16). Moreover, over the last three decades, a number of PAH-specific treatments have been approved, all of which address endothelial dysfunction and vasoconstriction rather than complex arterial remodeling (17). While these vasodilator agents can improve functional capacity, pulmonary hemodynamics and reduce hospitalization (18), their effect on survival is less clear of despite all available therapy, PAH, still carries a poor outlook, eventually leading to right heart failure and death with a 5-year survival of little more than 50% in a recent study (17), underlining the need for novel therapeutic approaches. Although several studies have elucidated the importance of endothelial senescence in atherosclerosis, heart failure, and metabolic disorders, its role in the pathogenesis of PAH is unclear (19). Consequently, gaining insight into the role of endothelial senescence in this disease may pave the way for innovative therapeutic approaches for the treatment of this otherwise progressive and often lethal condition.

Cellular senescence refers to a state of permanent growth arrest induced by stress as was initially described in cultured fibroblasts (15) Unfortunately, there is no single marker for identifying senescent cells (SCs). A SC is typically characterized by its enlarged, flattened appearance, vacuolized cellular morphology, elevated cytoplasm-to-nucleus ratio, and occasionally, the presence of multiple nuclei (6). These cells exhibit high expression of cyclin-dependent kinase inhibitors, such as p21Cip1 and p16INK4a, and the accumulation of the lysosomal enzyme senescence-associated-β-galactosidase (SA β-gal). SCs characteristically secrete a variety of inflammatory cytokines, chemokines, growth factors and proteases, termed senescence-associated secretory phenotype (SASP), which can contribute to a proinflammatory micro-environment (7). Senescence reprogramming is not a static cell fate; rather, it is a dynamic, multi-step process characterized by distinct molecular signatures determined by heterochromatin rearrangement and metabolic changes (15). Cellular senescence can be induced by a wide variety of noxious stimuli, including persistent DNA damage, oxidative stress, mitochondrial dysfunction, oncogene activation (leading to oncogene-induced senescence), and telomere shortening (7).

Cellular senescence is not always pathological, and it has recently been recognized to be adaptive in a range of physiological processes, including embryonic development, cellular reprogramming, and responses to tissue injury, such as wound healing and tissue repair. Exiting the cell cycle, as opposed to undergoing apoptosis, offers physiological advantages, and the release of chemokines as part the SASP, such as colony stimulating factor 1 (CSF1), CCL2 and IL-8, serves as a signal for immunosurveillance to promote self-clearance of senescent cells and prevent a chronic inflammatory response (20). In contrast, senescent phenotype can also play an important role in age-related diseases, including chronic cardiovascular and lung diseases, such as atherosclerosis (21) and pulmonary fibrosis (15, 22). Indeed, the accumulation of SCs in tissues can hinder repair while, at the same time, foster both paracrine and endocrine inflammation through release of the bioactive SASP (19). A three-step multi-marker method has been suggested to enhance the precision of identifying SCs involving: 1) the initial screening for senescence using markers (e.g., SA-β-gal or lipofuscin accumulation); 2) subsequent co-staining for the expression of other genes commonly found in SCs (e.g., P16, P21) or the absence of proliferation markers (e.g. Ki67 and Lamin B1); and 3) the identification of factors associated cellular senescence in a specific context, including the presence of SASP, evidence of DNA damage and senescence associated-signaling pathways such as PI3K/FOXO/mTOR as illustrated in Figure 1 (23). Importantly, expression of each characteristic of cellular senescence depends on the context, and these can differ depending on the nature of the stress trigger, the specific cell or tissue type, and the time elapsed since the initiation of the senescence program (24).

The endothelium is a dynamic cell monolayer representing the inner lining all blood vessels and playing an essential role in vascular development and the control of vascular tone and structure, while providing a thromboresistant surface to maintain blood fluidity and selective vascular permeability (19). The endothelium is highly heterogeneous, with profound differences in its structure and function in different tissue beds and in different regions of the vasculature (25). ECs are continuously exposed to shear forces related to blood flow and a wide variety of circulating factors, both physiological and pathological. Under normal conditions, the release of endothelial factors such as NO, plays a vital role in maintaining appropriate vascular tone and inhibiting platelet adhesion and activation (26). However, in pathological states, the endothelium is often the initial target for vascular injury (19), resulting in impaired tissue and organ function by disrupting blood flow and barrier function. Additionally, ‘activated’ ECs can release vasoconstrictor factors, such as endothelin-1 (27), and pro-inflammatory cytokines and chemokines (28) which can transmit damaging signals to other vascular and non-vascular cells and tissues. Sustained endothelial damage can also result in EC senescence, which has been implicated in a wide variety of vascular diseases (15). Endothelial senescence has been observed in different vascular regions, including the kidney, retina, liver, brain, lung, and aorta, consistent with a role in numerous pathological processes linked to vascular dysfunction (19). Several studies have emphasized the significance of endothelial senescence, especially in the context of pulmonary diseases. Amsellem et al. reported an elevated percentage of senescent lung ECs in individuals with chronic obstructive pulmonary disease, driven by telomere erosion and associated with increased inflammatory signaling mediated by IL-6, IL-8, MCP1 (monocyte chemotactic protein 1), and sICAM-1 (soluble intercellular adhesion molecule 1) (29). Senescent ECs have also been shown to play a detrimental role in the progression of pulmonary fibrosis (30), potentially by promoting endothelial to mesenchymal transition (EndMT) and enhancing the myofibroblastic transition of resident lung fibroblasts. This study suggested that targeting senescent ECs could be a promising pharmacotherapeutic approach for preventing and treating idiopathic pulmonary fibrosis (IPF), particularly in the elderly population. As well, it was recently reported that cellular senescence plays a role in the progression of bronchopulmonary dysplasia (BPD) (31), a disease of arrested lung development in extremely premature infants (32). This was evidenced by an increase in cellular senescence markers in the type-2 alveolar epithelial cells (AT2) and ECs in both a rat model and human BPD lungs, including elevated levels of oxidative DNA damage, tumor suppressors, GL-13, and inflammatory cytokines, along with reduced cell proliferation and lamin B expression. Administration of DRI-FOXO4, a senolytic agent that antagonizes Foxo4 binding to p53, improved lung development, suggesting that this may be a novel treatment strategy for BPD (31, 33). Therefore, as in these conditions the release of inflammatory cytokines and part of the SASP could play an important role in propagated vascular inflammation and promoting lung arterial remodeling on PAH. Table 1 summarizes the expression of endothelial senescence markers in pulmonary hypertension.

EC senescence in PH: One of the first reports implicating cellular senescence in PH demonstrated that impaired iron-sulfur biogenesis, resulting from frataxin deficiency, contributed to pulmonary vascular EC senescence that was associated with PH development (36). Frataxin is a mitochondrial protein and iron chaperone that plays a critical role in the formation of iron-sulfur (Fe-S) clusters (38). Reduced frataxin expression was also found to be correlated with increased p16INK4 in lung sections from patients with both PAH and PH associated with lung disease (Group 3 PH), as well as in animal models of PAH and Group 2 PH. A study using in situ lung specimens revealed that patients with chronic obstructive pulmonary disease (COPD) showed a higher percentage of senescent pulmonary endothelial cells by p16 and p21 staining compared to control subjects (35). Noureddine et al. (29) also reported that the percentage of senescent ECs, was higher in pulmonary vessels from patients with COPD compared to controls and positively correlated with the wall thickness area ratio (29). Wang et al. (34) demonstrated that pulmonary arterial ECs (PAECs) derived from patients with idiopathic PAH, exhibited significantly elevated expression of the senescence marker p53, which correlated with increased Bax/Bcl-2 levels compared to normal controls (34). Intriguingly, the use of a senolytic agent effectively reversed PH in these preclinical models, suggesting that targeting cellular senescence could provide a potential treatment for PH, regardless of the specific etiology (36, 39).

A recent study examined the mechanisms by which EC senescence may lead to hypoxia-induced PH using endothelial-specific progeroid mice that exhibit selectively EC aging and senescence (40). Chronic exposure to hypoxia is a well-established model that results in mild to moderate PH associated with abnormal pulmonary arterial muscularization, but not more complex arterial remodeling that is typically seen in PAH. Progeroid mice overexpress the dominant-negative form of telomeric repeat-binding factor 2 (TRF2DN) in endothelial cells under the control of the Tie2 or vascular endothelial cadherin promoter (41, 42). EC progeroid mice developed exaggerated PH in response to chronic hypoxia with greater pulmonary arterial medial remodeling compared to wild-type mice. Mechanistically, they found that progeroid mice exhibited increased endothelial expression of Notch ligands, leading to heightened Notch signaling, which promoted proliferation and migration in neighboring SMCs. Moreover, pharmacological inhibition of Notch signaling using the Gamma-Secretase inhibitor, DAPT, blocked the proliferative and pro-migratory effects of co-culture of SMCs with senescent ECs in-vitro and reduced PH in response to hypoxia in-vivo both in progeroid mice and wildtype mice. These findings support a disease-modifying role of EC senescence in PH and point to enhanced Notch signaling as potential pharmacotherapeutic target (40). However, further investigations are needed to fully define the extent of aberrant senescent-specific crosstalk that contributes to pulmonary vascular remodeling. Of note, the composition of SASP profiles varies based on the specific disease context (43). Consequently, future studies are needed to elucidate the unique SASP profile of senescent pulmonary ECs in PAH and how endothelial senescence-related signaling precisely regulates the recruitment of immune cells.

Given the exuberant nature of the arterial pathology in PAH, which includes a profound inflammatory response, it is tempting to speculate that cellular senescence plays a key role in promoting complex arterial remodeling ( Figure 2 ). While EC injury/apoptosis appears to be the key triggers for this condition, it is likely that other ‘hits’ are needed for the development of progressive disease. The study of underlying mechanisms of disease is greatly facilitated by the availability of experimental models that can faithfully reproduce its main clinical features. For many years, studies into the underlying mechanisms of PAH were limited mainly to chronic hypoxia (CH) and monocrotaline (MCT) models, neither of which fully reproduced the complex lung arterial pathology of PAH (44). CH is a model of group 3 PH due to lung disease whereas MCT is a plant-derived alkaloid that is toxic to pulmonary vascular ECs (45), but also has off target toxicity that can limit its usefulness, and again does not typically produce occlusive arterial remodeling. Perhaps the most relevant model was developed by inhibiting the VEGF receptor using a tyrosine kinase receptor antagonist, SU5416 (SU or Semaxanib). A single subcutaneous injection of SU, together with a 3-week exposure to CH, resulted in widespread lung EC apoptosis and reactive vascular cell proliferation, and a severe and progressive PAH phenotype, faithfully reproducing the hallmark lung vascular pathological features, including the complex arterial lesions (46). Recently, we have shown that perturbed blood flow, resulting in pathological levels of intimal shear stress, induces ongoing endothelial injury and apoptosis in this model, as the driver of complex and occlusive arterial remodeling (46, 47). Indeed, using unilateral pulmonary artery banding to hemodynamically offload the left lung, Abe et al. showed that ongoing hemodynamic perturbations was required for the development of complex plexiform-like lesions in an experimental model of severe PAH (46). This is analogous to the mechanism of PAH development in patients PAH associated with CHD due to large systemic to pulmonary vascular shunts, ultimately resulting in Eisenmenger’s syndrome (48, 49). In this context, excessive pulmonary blood flow causes ongoing endothelial injury, setting the stage for development of EC senescence by a variety of mechanisms implicated in endothelial dysfunction, including increased oxidative stress (15), DNA damage (50, 51) and mitochondrial dysfunction (52) ( Figure 2 ). It is also possible that ongoing EC injury may overwhelm endothelial repair mechanisms, leading to proliferative exhaustion and senescent changes in regenerative cells.

The importance cellular senescence in various pathological settings has made this an attractive therapeutic target for a wide range of diseases (53). Due to the absence of a precise biomarker for targeted treatment, current approaches to SC-directed therapy primarily focus on three key strategies: 1) disrupting crucial pathways that mediate senescence, such as pro-survival signaling; 2) targeting specific factors of the SASP; and (3) enhancing clearance of SCs by the immune system (20, 53).

There is great enthusiasm for the development of treatments that target senescent cells across a wide variety of diseases. However, the lack of a universal senescence biomarker poses challenges for translational efforts, necessitating detailed characterization of SCs in different disease contexts. Determining when and how to target SCs is crucial due to the pleomorphic and dynamic nature of biological effects on cellular senescence, requiring in depth understanding of senescence responses in specific settings to identify optimal therapeutic strategies and windows, highlighting the need for comprehensive phenotypic characterization to inform treatment design. The role of EC senescence in PAH is still poorly defined, necessitating careful assessment of the biology of cellular senescence in preclinical models to determine optimal treatment strategies and timing. Conflicting outcomes with the use of senolytic therapies in preclinical models of PAH necessitate further investigations to identify the specific characteristics of senescent ECs and define the spatial and temporal factors that determine their biological effects during disease evolution. Utilizing advanced technologies, such as single-cell transcriptomics and proteomics, will be instrumental in identifying which specific cells undergo senescence in PAH and defining at high resolution the spatiotemporal sequences of senescent changes during disease evolution. This detailed cellular and mapping could inform the development of more effective senotherapies by allowing for more precise targeting of senescent cells. As well, more tailored strategies, such as cell-type-specific or targeted immunotherapeutic approaches, will be essential in the design of the next generation treatments.

ESQ: Writing – original draft. DS: Writing – review & editing.